The invention is a process for removing bacterial endotoxin from gram negative polysaccharides.
Bacterial endotoxin is a potent pyrogen that can often produce fever reactions when administered to patients. Endotoxin is an integral component of the outer cell surface of Gram-negative bacteria. It exists in its natural state as a complex with lipid, carbohydrate and protein. When highly purified, endotoxin does not contain protein, and by its chemical composition is referred to as a lipopolysaccharide (see Weary and Pearson, Bio. Pharm. April (1988) pp. 22-29).
The outer-wall layer of Gram-negative bacteria serves as an outer barrier through which materials must penetrate if they are to reach the cell; it is selectively permeable. Generally, endotoxin is released in large amounts only when the cell wall is lysed.
Removal of contaminating endotoxin from Gram-negative polysaccharides is important when the polysaccharide is to be administered to humans. Endotoxins in large quantities can cause shock, severe diarrhea, fever and leukopenia followed by leukocytosis, and can elicit the Shwartzman and Sanarelli-Shwartzman phenomena.
U.S. Pat. No. 4,695,624 describes covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of these polysaccharides with immunogenic proteins, and methods of preparing the polysaccharides and conjugates and of confirming covalency. The patent describes purification of the polysaccharide in Example 1, beginning in column 14. After fermentation, inactivation and cell removal, the resulting product undergoes a series of cold ethanol fractionations. Following phenol extraction are diafiltration, ethanol precipitation, ultracentrifugation in ethanol, and collection of the finished product.
Frequently, the amount of contaminating endotoxin remaining after the above-described procedure is higher than desired.
Methods for removing endotoxin which are known in the art are described by Weary and Pearson (ibid): rinsing with nonpyrogenic solution (Feldstine et al., J. Parenter. Drug Assoc., 33, p. 125 (1979) and Berman et al., J. Parenter. Sci. Technol., 41, p. 158 (1987); distillation; ultrafiltration using membranes rated by molecular weight exclusion (Sweadner et al., Appl. Environ. Microbiol., 34, p. 382 (1977) and Henderson et al., Kidney Int., 14, p. 522 (1978); reverse osmosis using thin cellulose acetate or polyamide materials (Nelson, Pharm. Technol., 2, p. 46 (1978); electrostatic attraction (Gerba et al., Pharm Technol., 4, p. 83 (1980) and Hou et al., Appl. Environ. Microbiol., 40, p. 892 (1980); hydrophobic attraction using aliphatic polymers (Robinson et al., in Depyrogenation (Parental Drug Association, Philadelphia (1985), pp. 54-69); adsorption using activated carbon (Berger et al., Adv. Chem. Ser., 16, p. 169 (1956), Gemmell et al., Pharm J., 154, p. 126 (1945), and Brindle et al., Pharm J., 157, p. 85 (1946); and affinity chromatography (Soter, Bio/Technology, 12, p. 1035 (1984).
Sawada, et al., Applied and Environmental Microbiology, April 1986, pp. 813-820, describe removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane. Gerba et al., Applied and Environmental Microbiology, December 1985, pp. 1375-1377, describe endotoxin removal from various solutions using charged nylon and cellulose-diatomaceous earth filters. Nolan et al., Proceedings of the Society for Experimental Biology and Medicine, Vol. 149, pp. 766-770 (1975), describe endotoxin binding by charged and uncharged resins.
It is a purpose of the present invention to provide an effective, accurate method for obtaining Gram-negative polysaccharide mixtures having low or negligable levels of endotoxin.